The ability to assess the expression of multiple genes in individual cells represents a powerful tool for studying the messenger RNA (mRNA) abundances within identifiable cell types. One such technique, the amplified antisense RNA (aRNA) method (Eberwine, J. et al. 1992 Proc. Natl. Acad. Sci. USA 89:3010-3014) allows the simultaneous identification of relative messenger RNA (mRNA) levels for multiple genes within single cells. The first introduction of in situ transcription (IST) allowed the analysis of gene expression within fixed tissue sections (Tecott, L. H. et al. 1988 Science 240:1661-1664). The recent development of the aRNA procedure coupled with IST permitted the analysis of the relative levels of multiple mRNAs within single, dissociated cells (Eberwine, J. et al. 1992 Proc. Natl. Acad. Sci. USA 89:3010-3014). In addition, the aRNA technique can be combined with electrophysiological recordings from dissociated cells (Eberwine, J. et al. 1992 Proc. Natl. Acad. Sci. USA 89:3010-3014) or cells from slice preparations (Mackler, S. A. et al. 1992 Neuron 9:539-548) to provide a functional correlate of gene expression changes. The cellular specificity of aRNA amplification offers a distinct advantage over other techniques used to evaluate gene expression. For example, Northern analysis involves extraction of RNA from tissue homogenates which include a heterogeneous population of cells particularly within the central nervous system (CNS). In situ hybridization can be used to study gene expression in individual cells, but the study of multiple genes is complex. Another method, PCR, limits analysis to only a few genes at one time (Eberwine, J. et al. 1995 The Neuroscientist 1:200-211).
Recently, the aRNA procedure has been extended to characterize the expression of mRNA abundances for multiple genes within immunohistochemically labeled cells (Crino, P. B. et al. 1996 Proc. Natl. Acad. Sci. USA 93:14152-14157). This method allows additional phenotypic characterization of cells prior to single cell amplification. While this method of cell identification is useful for identifying cells based on the expression of a particular protein, imamunohistochemical detection is problematic when applied to analyzing the molecular changes in degenerating or dying cells. Specifically, a frequent hallmark of damaged cells is disruption of protein turnover. Therefore, particular proteins may be decreased or even absent in dying cells. Such alterations in protein expression and activity have been reported for a variety of CNS insults (Ferrer, I. et al. 1993 Clin. Neuropath. 12:53-58; Taft, W. C. et al. 1993 J. Cereb. Blood Flow Metab. 13:796-802; Hicks, R. R. et al. 1996 Acta Neuropathol. 91:236-246). However, other proteins are upregulated in neurologic disease (Anderson, A. J. et al. 1994 Exp. Neurol. 125:286-295). Thus, the expression of immunohistochemical markers may reveal abnormal cell populations; or alternatively, the expression of certain proteins in cells may be associated with cell death. Without a specific marker of cell damage, however, positive identification of damaged/dying cells based on immunohistochemical criteria is unreliable.
Methods for identifying dying cells by DNA damage stains have been described. For example, terminal deoxynucleotidyl-transferase (Tdt) mediated biotin-dUTP nick end labeling (TUNEL) technique has been used to identify dying cells in a developing brain (Gavrieli et al. 1992 J. Cell Biol. 119:493-501). The TUNEL stain utilizes the enzyme Tdt which incorporates biotinylated nucleotides to the 3' ends of fragmented DNA and has been used as a marker for dying cells. This method is useful for identifying cells that are undergoing programmed cell death (PCD), a phenomenon which occurs as a consequence of normal development (Oppenheim, R. W. 1991 Annual Review of Neuroscience 14:453-501). TUNEL-positive cells have also been found in pathological conditions including traumatic brain injury (Rink, A. et al. 1995 Am. J. Pathol. 147:1575-1583; Colicos, M. A. and Dash, P. K. 1996 Brain Research 739:102-131; Conti, A. C. et al. 1996 J. Neurotrauma 13:595), ischemia (Li, Y. et al. 1995 Stroke 26:1252-1257), tumors (Ikeda, H. et al. 1996 Am. J. Surg. Pathol. 20:649-655), Alzheimer's disease (Smale, G. et al. 1995 Exp. Neurol. 133:225-230; Tronsco, J. C. et al. 1996 J. Neuropathol. Exp. Neurol. 55:1134-1142), Parkinson's disease (Mochizuki, H. et al. 1997 J. Neural. Transm. Suppl. 50:125-140), Huntington's disease (Thomas, L. B. et al. 1995 Exp. Neurol. 133:265-272), multiple sclerosis (Dowling, P. et al. 1996 J. Exp. Med. 184:1513-1518) and amyotrophic lateral sclerosis (ALS); Troost, D. et al., 1995 Neuropathol. and Applied Neurobiol. 21:498-504.